This invention relates to bacterial diagnostics, and more particularly to detection of Legionella species, particularly Legionella pneumophila. 
The genus Legionella, family Legionellaceae, includes over 40 different species of fastidious gram-negative bacilli, with over 60 described serogroups. While some of these organisms represent normal environmental flora, many have been shown to cause human disease, namely opportunistic pneumonia in immunocompromised patients. The vast majority of such cases (approximately 85%) are due to L. pneumophila, with the remainder due to other species, most commonly L. micdadei, L. bozemanii, L. dumoffii, and L. longbeachae. Legionella pneumonia can be community acquired or nosocomial, and sporadic or epidemic in nature. Pulmonary infection may be subclinical, or severe and life threatening. The fatality rate can approach 50% in immunocompromised patients. The organism often responds to antimicrobial therapy, usually with macrolides, and clinical responses usually occur within 3-5 days. The latter fact, combined with clinical and radiographic features that are often non-specific, serve to underscore the value of a prompt and accurate laboratory diagnosis.
The invention provides for methods of identifying Legionella in a biological sample, and further, for specifically detecting Legionella pneumophila. Primers and probes for detecting Legionella, specifically L. pneumophila, are provided by the invention, as are kits containing such primers and probes. Methods of the invention can be used to rapidly identify Legionella nucleic acids from specimens for diagnosis of Legionella infection. Using specific primers and probes, the methods include amplifying and monitoring the development of specific amplification products using real-time PCR.
In one aspect of the invention, there is provided a method for detecting the presence or absence of Legionella in a biological sample from an individual. The method to detect Legionella includes performing at least one cycling step, which includes an amplifying step and a hybridizing step. The amplifying step includes contacting the sample with a pair of 5S rRNA primers to produce a 5S rRNA amplification product if Legionella nucleic acid encoding 5S rRNA is present in the sample, and the hybridizing step includes contacting the sample with a pair of 5S rRNA probes. Generally, the members of the pair of 5S rRNA probes hybridizes to the amplification product within no more than five nucleotides of each other. A first 5S rRNA probe of the pair of 5 S rRNA probes is typically labeled with a donor fluorescent moiety and a second 5S rRNA probe of the pair of 5S rRNA probes is typically labeled with a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of fluorescent resonance energy transfer (FRET) between the donor fluorescent moiety of the first 5S rRNA probe and the acceptor fluorescent moiety of the second 5S rRNA probe. The presence of FRET is usually indicative of the presence of Legionella in the biological sample, while the absence of FRET is usually indicative of the absence of Legionella in the biological sample. In addition, determining the melting temperature between one or both of the 5S rRNA probe(s) and the corresponding probe targets can confirm the presence or absence of the Legionella.
In another aspect, the invention features a method for detecting the presence or absence of L. pneumophila in a biological sample from an individual. The method to detect L. pneumophila includes performing at least one cycling step, which includes an amplifying step and a hybridizing step. The amplifying step includes contacting the sample with a pair of mip primers to produce a mip amplification product if L. pneumophila nucleic acid encoding mip is present in the sample, and the hybridizing step includes contacting the sample with a pair of mip probes. Generally, the members of the pair of mip probes hybridizes to the amplification product within no more than five nucleotides of each other. A first mip probe of the pair of mip probes is typically labeled with a donor fluorescent moiety and a second mip probe of the pair of mip probes is typically labeled with a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of FRET between the donor fluorescent moiety of the first mip probe and the acceptor fluorescent moiety of the second mip probe. The presence of FRET is usually indicative of the presence of L. pneumophila in the biological sample, while the absence of FRET is usually indicative of the absence of L. pneumophila in the biological sample. The method to detect L. pneumophila can be performed after the method has been performed to detect Legionella or concurrent with the method to detect Legionella.
A pair of 5S rRNA primers generally includes a first 5S rRNA primer and a second 5S rRNA primer. The first 5 S rRNA primer can include the sequence 5xe2x80x2-ACT ATA GCG ATT TGG AAC C-3xe2x80x2 (SEQ ID NO: 1), and the second 5S rRNA primer can include the sequence 5xe2x80x2-GGC GAT GAC CTA CTT TC-3xe2x80x2 (SEQ ID NO:2). The first 5S rRNA probe can include the sequence 5xe2x80x2-CAT GAG GAA GCC TCA CAC TAT CA-3xe2x80x2 (SEQ ID NO:3), and the second 5S rRNA probe can include the sequence 5xe2x80x2-GGC GAT GAC CTA CTT TC-3xe2x80x2 (SEQ ID NO:2). In certain aspects, the second 5S rRNA primer can be labeled with a donor fluorescent moiety and can act as the second 5S rRNA probe.
A pair of mip primers generally includes a first mip primer and a second mip primer. The first mip primer can include the sequence 5xe2x80x2-ACC GAA CAG CAA ATG AAA GA-3xe2x80x2 (SEQ ID NO:4), and the second mip primer can include the sequence 5xe2x80x2-AAC GCC TGG CTT GTT TTT GT-3xe2x80x2 (SEQ ID NO:5). The first mip probe can include the sequence 5xe2x80x2-AAC AAG TTT CAG AAA GAT TTG ATG GCA AAG-3xe2x80x2 (SEQ ID NO:6), and the second mip probe can include the sequence 5xe2x80x2-GTA CTG CTG AAT TCA ATA AGT AAG CGG ATG-3xe2x80x2 (SEQ ID NO:7).
The members of the pair of 5S rRNA probes can hybridize within no more than two nucleotides of each other, or can hybridize within no more than one nucleotide of each other. A representative donor fluorescent moiety is fluorescein, and representative acceptor fluorescent moieties include LC(trademark)-RED 640 (LightCycler(trademark)-Red 640-N-hydroxysuccinimide ester), LC(trademark)-RED 705 (LightCycler(trademark)-Red 705-Phosphoramidite), and cyanine dyes such as CY5 and CY5.5.
In one aspect, the detecting step includes exciting the biological sample at a wavelength absorbed by the donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by the acceptor fluorescent moiety. In another aspect, the detecting includes quantitating the FRET. In yet another aspect, the detecting step is performed after each cycling step, and further, can be performed in real-time.
Generally, the presence of the FRET in an amount at least 3 times the amount of FRET in a sample lacking the Legionella 5S rRNA nucleic acid molecule indicates the presence of a Legionella infection in the individual. Representative biological sample include sputum, bronchio-alveolar lavage, bronchial aspirates, lung tissue, urine and blood.
The above-described methods can further include preventing amplification of a contaminant nucleic acid. Preventing amplification can include performing the amplifying step in the presence of uracil and treating the biological sample with uracil-DNA glycosylase prior to a first amplification step. In addition, the cycling step can be performed on a control sample. A control sample can include a portion of the Legionella nucleic acid molecule encoding 5S rRNA. Alternatively, such a control sample can be amplified using a pair of control primers and hybridized using a pair of control probes. The control primers and the control probes are usually other than the 5S rRNA primers and 5S rRNA probes, respectively. A control amplification product is produced if control template is present in the sample, and the control probes hybridize to the control amplification product.
In another aspect of the invention, there are provided articles of manufacture, including a pair of 5S rRNA primers; a pair of 5S rRNA probes; and a donor fluorescent moiety and a corresponding fluorescent moiety. A pair of 5S rRNA primers generally includes a first 5S rRNA primer and a second 5S rRNA primer. The first 5S rRNA primer can include the sequence 5xe2x80x2-ACT ATA GCG ATT TGG AAC C-3xe2x80x2 (SEQ ID NO:1), and the second 5S rRNA primer can include the sequence 5xe2x80x2-GGC GAT GAC CTA CTT TC-3xe2x80x2 (SEQ ID NO:2). A pair of 5S rRNA probes generally includes a first 5S rRNA probe and a second 5S rRNA probe. The first 5S rRNA probe can include the sequence 5xe2x80x2-CAT GAG GAA GCC TCA CAC TAT CA-3xe2x80x2 (SEQ ID NO:3), and the second 5S rRNA probe can include the sequence 5xe2x80x2-GGC GAT GAC CTA CTT TC-3xe2x80x2 (SEQ ID NO:2). Articles of manufacture of the invention can further or alternatively include a pair of mip primers; a pair of mip probes; and a donor fluorescent moiety and a corresponding fluorescent moiety. A pair of mip primers generally includes a first mip primer and a second mip primer. The first mip primer can include the sequence 5xe2x80x2-ACC GAA CAG CAA ATG AAA GA-3xe2x80x2 (SEQ ID NO:4), and the second mip primer can include the sequence 5xe2x80x2-AAC GCC TGG CTT GTT TTT GT-3xe2x80x2 (SEQ ID NO:5). A pair of mip probes generally includes a first mip probe and a second mip probe. The first mip probe can include the sequence 5xe2x80x2-AAC AAG TTT CAG AAA GAT TTG ATG GCA AAG-3xe2x80x2 (SEQ ID NO:6), and the second mip probe can include the sequence 5xe2x80x2-GTA CTG CTG AAT TCA ATA AGT AAG CGG ATG-3xe2x80x2 (SEQ ID NO:7). The probes in such articles of manufacture can be labeled with a donor fluorescent moiety and with a corresponding acceptor fluorescent moiety. The article of manufacture can also include a package label or package insert having instructions thereon for using the pair(s) of primers and pair(s) of probes to detect the presence or absence of Legionella or L. pneumophila in a biological sample.
In yet another aspect, the invention provides a method for detecting the presence or absence of Legionella in a biological sample from an individual. Such a method includes performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step. An amplifying step includes contacting the sample with a pair of 5S rRNA primers to produce a 5S rRNA amplification product if a Legionella nucleic acid molecule encoding the 5S rRNA is present in the sample. A hybridizing step includes contacting the sample with a 5S rRNA probe, wherein the 5S rRNA probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety of the 5S rRNA probe. The presence or absence of FRET is indicative of the presence or absence of Legionella in the sample. Amplification can employ a polymerase enzyme having 5xe2x80x2 to 3xe2x80x2 exonuclease activity, and the donor and acceptor fluorescent moieties can be within no more than 5 nucleotides of each other on the probe. In such a method, the 5S rRNA probe can include a nucleic acid sequence that permits secondary structure formation that results in spatial proximity between the donor and the acceptor fluorescent moiety. In the above-described methods, the acceptor fluorescent moiety can be a quencher. A similar method can be used to detect L. pneumophila using a pair of mip primers and a mip probe.
In another aspect, the invention provides a method for detecting the presence or absence of Legionella in a biological sample from an individual. Such a method includes performing at least one cycling step, wherein a cycling step comprises an amplifying step and a dye-binding step. An amplifying step includes contacting the sample with a pair of 5S rRNA primers to produce a 5S rRNA amplification product if a Legionella nucleic acid molecule encoding a 5S rRNA is present in the sample. A dye-binding step comprises contacting the 5S rRNA amplification product with a nucleic acid binding dye. The method further includes detecting the presence or absence of binding of the nucleic acid binding dye to the amplification product. The presence of binding is usually indicative of the presence of Legionella in the sample, and the absence of binding is usually indicative of the absence of Legionella in the sample. Representative nucleic acid binding dyes include SYBRGREENI(copyright), SYBRGOLD(copyright), and ethidium bromide. Such a method can further include determining the melting temperature between the 5S rRNA amplification product and the nucleic acid binding dye. The melting temperature can confirm the presence or absence of the Legionella. Similarly, such a method can be used to specifically detect L. pneumophila using a pair of mip primers.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims.
The present invention provides for methods of detecting Legionella in a biological sample, and for specifically detecting L. pneumophila. Primers and probes for detecting Legionella infections in general, or specifically L. pneumophila infections, are provided. Articles of manufacture containing such primers and probes also are provided by the invention. With conventional culture serving as the xe2x80x9cgold standard,xe2x80x9d a real-time PCR assay was compared to a direct fluorescent antibody (DFA) assay for the detection of Legionella species in BAL specimens, and to a DFA assay, in situ hybridization (ISH), and Warthin Starry (WS) staining for the detection of Legionella species in open lung biopsy specimens. The increased sensitivity of real-time PCR compared to other methods, as well as the improved features of real-time PCR including sample containment and real-time detection of the amplified product indicate the feasibility for implementation of this technology for routine diagnosis of Legionella infections, specifically those attributable to L. pneumophila, in the clinical laboratory.
Legionella Species and L. pneumophila 
Bacteria of the genus Legionella are intracellular parasites and major human pathogens. They bind to surface receptors, penetrate eukaryotic cells and initiate complex disorders during phagocytosis. These disorders include inhibition of oxidative burst, a decrease in phagosome acidification, the blocking of phagasome maturation and changes in organelle trafficking. As a result, the microorganisms prevent the bactericidal activity of the phagocyte and transform the phagosome into a niche for their replication. Biological, biochemical and molecular-genetic approaches have been used to identify a panel of bacterial products that may be involved in Legionella virulence. They include cytotoxins, several enzymes and a set of genes thought to encode proteins of the export machinery. The interaction of virulent Legionella with phagocytic cells can be arbitrarily divided into several steps: binding of microorganisms to receptors on the surface of eukaryotic cells, penetration of microorganisms into phagocytes, escape from bactericidal attack, formation of a replicative vacuole, intracellular multiplication, and killing of the host cell.
In many bacteria, the 16S, 23S, and 5S rRNAs are encoded in unlinked operons approximately 5 kilobases (kb) in length. E. coli and S. typhimurium have seven rRNA operons. When multiple operons are present, they are organized similarly and have the order 16S, 23S and 5S. Genes for tRNA are generally located between the 16S and 23S rRNA genes and sometimes after the 5S gene. The three mature rRNA sequences are separated by spacer sequences that are removed during processing. The spacer regions are highly conserved between operons. Long inverted repeats, flanking both the 16S and 23S rRNAs, have the potential to form double-stranded stems at the base of the 16S and 23S rRNAs. These double-stranded regions are predicted to be stable in vivo and have been observed directly by electron microscopy. Each operon contains two tandem promoters, one of which is responsive to control by guanosine 5xe2x80x2-diphosphate, 3xe2x80x2-diphosphate (ppGpp), and the other that is subject to growth rate control. Initial cleavages separate 16S and 23S RNA, usually before transcription of the operon is complete.
Mutations in a gene coding for a 24 kDa surface protein of Legionella species, as well as other intracellular organisms such as Chlamydia, Coxiella and Rickettsia, result in a severe reduction in virulence towards macrophages, macrophage-like cell lines, alveolar epithelial cells and protozoa. They also cause considerable attenuation of L. pneumophila in laboratory animals. As such mutants are impaired in their ability to initiate macrophage infection, the mutated surface component was named the macrophage infectivity potentiator (mip) protein.
The deduced amino acid sequence of the mip protein from L. pneumophila shows homology to human, Neurospora and yeast proteins able to bind the immunosupressant drug FK506. FK506-binding proteins are receptors belonging to a family of peptidyl-prolyl cisltrans isomerases (PPIs) called immunophilins, which catalyze the cisltrans interconversion of prolyl imidic peptide bonds in proteins. Investigations with the 24 kDa mip protein confirmed that it has isomerase activity. In addition, the inhibitory effect of FK506 on mip was similar to that on human FK506-binding protein.
The N-terminus of mip, which is predicted to be a 60-amino acid xcex1-helix, apparently anchors the protein to the bacterial cell wall. The C-terminus, which carries a domain possessing peptidyl-prolyl cisltrans isomerase, projects distally from the bacterial surface to accomplish its biological function. Data from an X-ray solution scattering study suggested that the mip protein functions as a dimer.
Legionella Nucleic Acids and Oligonucleotides
The invention provides methods to detect Legionella by amplifying Legionella nucleic acid molecules encoding, for example, a portion of the 5S rRNA. The invention further provides methods to specifically detect L. pneumophila by amplifying L. pneumophila nucleic acid molecules encoding, for example, mip. Legionella and L. pneumophila-specific nucleic acid molecules other than those exemplified herein (e.g., those encoding 5S rRNA and mip, respectively) can be used to detect Legionella and L. pneumophila in a sample and are known to those of skill in the art. Nucleic acid sequences encoding Legionella 5S rRNA have been described (see, for example, GenBank Accession Nos. Z30435 or Z30540), as have nucleic acid sequences encoding L. pneumophila mip (see, for example, GenBank Accession Nos. AF095230 and AF095220). Specifically, primers and probes to amplify and detect Legionella 5S rRNA nucleic acid molecules are provided by the invention. Similarly, primers and probes to amplify and detect L. pneumophila mip nucleic acid molecules are also provided by the invention.
Primers that amplify a Legionella nucleic acid molecule, e.g., a nucleic acid molecule encoding mip or a portion of the 5S rRNA, can be designed using, for example, a computer program such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Important features when designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of a pair of primers, and the length of each primer (e.g., the primers need to be long enough to anneal with sequence-specificity and to initiate synthesis but not so long that fidelity is reduced during oligonucleotide synthesis). Typically, oligonucleotide primers are 8 to 50 nucleotides in length (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length). xe2x80x9c5S rRNA primersxe2x80x9d as used herein refers to oligonucleotide primers that specifically anneal to Legionella nucleic acid sequences encoding 5S rRNA and initiate synthesis therefrom under appropriate conditions. Likewise, xe2x80x9cmip primersxe2x80x9d refers to oligonucleotide primers that specifically anneal to L. pneumophila nucleic acid sequences encoding mip and initiate synthesis therefrom under appropriate conditions.
Designing oligonucleotides to be used as hybridization probes can be performed in a manner similar to the design of primers, although the members of a pair of probes preferably hybridize to an amplification product within no more than 5 nucleotides of each other on the same strand such that fluorescent resonance energy transfer (FRET) can occur (e.g., within no more than 1, 2, 3, or 4 nucleotides of each other). This minimal degree of separation typically brings the respective fluorescent moieties into sufficient proximity such that FRET occurs. It is to be understood, however, that other separation distances (e.g., 6 or more nucleotides) are possible provided the fluorescent moieties are appropriately positioned relative to each other (for example, with a linker arm) such that FRET can occur. In addition, probes can be designed to hybridize to targets that contain a polymorphism or mutation, thereby allowing differential detection of Legionella species or members within a species based on either absolute hybridization of different pairs of probes corresponding to the particular Legionella species or member to be distinguished or differential melting temperatures between, for example, members of a pair of probes and each amplification product corresponding to the Legionella species or member to be distinguished (e.g., L. pneumophila from L. oakridgenesis). As with oligonucleotide primers, oligonucleotide probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are 8 to 50 nucleotides in length (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length). xe2x80x9c5S rRNA probesxe2x80x9d as used herein refers to oligonucleotide probes that specifically anneal to a 5S rRNA amplification product. Similarly, xe2x80x9cmip probesxe2x80x9d refers to oligonucleotide probes that specifically anneal to a mip amplification product.
Constructs of the invention include vectors containing Legionella nucleic acid molecules or fragments thereof, for example, those encoding 5S rRNA or mip. Constructs can be used, for example, as a control template nucleic acid. Vectors suitable for use in the present invention are commercially available and/or produced by recombinant DNA technology methods routine in the art. A Legionella nucleic acid molecule encoding 5S rRNA or mip can be obtained, for example, by chemical synthesis, direct cloning from a Legionella species, or by PCR amplification. A Legionella 5S rRNA or mip nucleic acid molecule, or fragments thereof, can be operably linked to a promoter or other regulatory element such as an enhancer sequence, a response element, or an inducible element that modulates expression of the 5S rRNA or mip nucleic acid molecule. As used herein, operably linking refers to connecting a promoter and/or other regulatory elements to a Legionella nucleic acid encoding a 5S rRNA or mip in such a way as to permit and/or regulate expression of the 5S rRNA or mip nucleic acid molecule. A promoter that does not normally direct expression of a 5S rRNA nucleic acid sequence can be used to direct transcription of a 5S rRNA nucleic acid molecule using, for example, a viral polymerase, a bacterial polymerase, or a eukaryotic RNA polymerase II. Alternatively, the 5S rRNA native xe2x80x9cinternalxe2x80x9d promoter can be used to direct transcription of a 5S rRNA nucleic acid using, for example, an RNA polymerase III enzyme. In addition, operably linked can refer to an appropriate connection between a Legionella 5S rRNA or mip promoter or other regulatory element to a heterologous coding sequence (e.g., a non-5S rRNA or non-mip coding sequence, for example, a reporter gene) in such a way as to permit expression of the heterologous coding sequence.
Constructs suitable for use in the methods of the invention typically include, in addition to Legionella nucleic acid molecules encoding 5S rRNA or mip, sequences encoding a selectable marker (e.g., an antibiotic resistance gene) for selecting desired constructs and/or transformants, and an origin of replication. The choice of vector systems usually depends upon several factors, including, but not limited to, the choice of host cells, replication efficiency, selectability, inducibility, and the ease of recovery.
Constructs of the invention containing Legionella nucleic acid molecules encoding 5S rRNA or mip can be propagated in a host cell. As used herein, the term host cell is meant to include prokaryotes and eukaryotes such as yeast, plant and animal cells. Prokaryotic hosts may include E. coli, Salmonella typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. A construct of the invention can be introduced into a host cell using any of the techniques commonly known to those of ordinary skill in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. In addition, naked DNA can be delivered directly to cells (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466).
Polymerase Chain Reaction (PCR)
U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically employs two primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in the present invention include oligonucleotides capable of acting as a point of initiation of nucleic acid synthesis within the Legionella 5S rRNA nucleic acid molecule or L pneumophila mip nucleic acid molecule. A primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically. The primer is preferably single-stranded for maximum efficiency in amplification, but a primer can be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double stranded nucleic acids is by heating.
The term xe2x80x9cthermostable polymerasexe2x80x9d refers to a polymerase enzyme that is heat stable, i.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessaryto effect denaturation of double-stranded template nucleic acids. Generally, the synthesis is initiated at the 3xe2x80x2 end of each primer and proceeds in the 5xe2x80x2 to 3xe2x80x2 direction along the template strand. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR provided the enzyme is replenished.
If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One method of separating thenucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditions necessary for denaturing template nucleic acid will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90xc2x0 C. to about 105xc2x0 C. for a time depending on features of the reaction such as temperature and the nucleic acid length.
If the double-stranded nucleic acid is denatured by heat, the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence on the template nucleic acid. The temperature for annealing is usually from about 35xc2x0 C. to about 65xc2x0 C. The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient for extension to occur from the annealed primer to generate products complementary to the template nucleic acid. The temperature should be sufficient to synthesize an extension product from each primer that is annealed to a nucleic acid template, but should not be so high as to denature an extension product from its complementary template. The temperature generally ranges from about 40xc2x0 to 80xc2x0 C.
PCR assays can employ, for example, DNA or RNA, including messenger RNA (mRNA). The template nucleic acid need not be purified; it may be a minor fraction of a complex mixture, such as Legionella nucleic acid contained in human cells. DNA or RNA may be extracted from any biological sample such as sputum, a bronchio-alveolar lavage, bronchial aspirates, lung tissue, urine or blood by routine techniques such as those described in Diagnostic Moleculear Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington D.C.). Template nucleic acids can be obtained from any number of sources, such as plasmids, or natural sources including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.
The oligonucleotide primers are combined with other PCR reagents under reaction conditions that induce primer extension. For example, chain extension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.001% (w/v) gelatin, 0.5-1.0 xcexcg denatured template DNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO). The reaction usually contains 150 to 320 xcexcM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.
The newly synthesized strands form a double-stranded molecule that can be used in the succeeding steps of the reaction. The steps of strand separation, annealing, and elongation can be repeated as often as needed to produce the desired quantity of amplification products corresponding to the target Legionella nucleic acid molecule. The limiting factors in the reaction are the amounts of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., amplification and hybridization) are preferably repeated at least once. For use in detection, the number of cycling steps will depend, e.g., on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps may be required to amplify the target sequence sufficient for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.
Fluorescent Resonance Energy Transfer (FRET)
FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the fact that when a donor and a corresponding acceptor fluorescent moiety are positioned within a certain distance of each other, energy transfer takes place between the two fluorescent moieties that can be visualized or otherwise detected and/or quantitated. As used herein, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to an amplification product at particular positions determined by the complementarity of the oligonucleotide probes to the Legionella target nucleic acid sequence. Upon hybridization of the oligonucleotide probes to the amplification product at the appropriate positions, a FRET signal is generated.
Fluorescent analysis can be carried out using, for example, a photon counting epifluorescent microscope system (containing the appropriate dichroic mirror and filters for monitoring fluorescent emission at the particular range), a photon counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer can be carried out with an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation in the desired range.
As used herein with respect to donor and corresponding acceptor fluorescent moieties, xe2x80x9ccorrespondingxe2x80x9d refers to an acceptor fluorescent moiety having an emission spectrum that overlaps the excitation spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety preferably should be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Accordingly, efficient non-radiative energy transfer can be produced therebetween.
Fluorescent donor and acceptor moieties are generally chosen for (a) high efficiency Fxc3x6rster energy transfer; (b) a large final Stokes shift ( greater than 100 nm); (c) shift of the emission as far as possible into the red portion of the visible spectrum ( greater than 600 nm); and (d) shift of the emission to a higher wavelength than the Raman water fluorescent emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be chosen that has its excitation maximum near a laser line (for example, Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, a high quantum yield, and a good overlap of its fluorescent emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be chosen that has a high extinction coefficient, a high quantum yield, a good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red part of the visible spectrum ( greater than 600 nm).
Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4xe2x80x2-isothio-cyanatostilbene-2,2xe2x80x2-disulfonic acid, 7-diethylamino-3-(4xe2x80x2-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1-pyrenebutyrate, and 4-acetamido-4xe2x80x2-isothiocyanatostilbene-2,2xe2x80x2-disulfonic acid derivatives. Representative acceptor fluorescent moieties, depending upon the donor fluorescent moiety used, include LC(trademark)-RED 640 (LightCycler(trademark)-Red 640-N-hydroxysuccinimide ester), LC(trademark)-RED 705 (LightCycler(trademark)-Red 705-Phosphoramidite), cyanine dyes such as CY5 and CY5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate or other chelates of Lanthanide ions (e.g., Europium, or Terbium). Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).
The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm also is important, as the linker arms will affect the distance between the donor fluorescent moiety and the acceptor fluorescent moiety. The length of a linker arm for the purpose of the present invention is the distance in Angstroms (xc3x85) from the nucleotide base to the fluorescent moiety. In general, a linker arm is from about 10 to about 25 xc3x85. The linker arm may be of the kind described in WO 84/03285. WO 84/03285 also discloses methods for attaching linker arms to particular nucleotide bases, and also for attaching fluorescent moieties to a linker arm.
An acceptor fluorescent moiety such as LC(trademark)-RED 640 (LightCycler(trademark)-Red 640-N-hydroxysuccinimide ester) can be combined with C6-Phosphoramidites (available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to produce, for example, LC(trademark)-RED 640 (LightCycler(trademark)-Red 640-Phosphoramidite). Frequently used linkers to couple a donor fluorescent moiety such as fluorescein to an oligonucleotide include thiourea linkers (FITC-derived, for example, fluorescein-CPG""s from Glen Research or ChemGene (Ashland. Mass)), amide-linkers (fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3xe2x80x2-amino-CPG""s that require coupling of a fluorescein-NHS-ester after oligonucleotide synthesis.
Detection of Legionella and L. pneumophila 
The diagnosis of Legionella infection can be made from a number of specimen types, and by a number of testing modalities. Bacterial culture of bronchoscopy or lung biopsy specimens is the most sensitive means of detection to date. Specialized growth media such as Buffered Charcoal Yeast Extract (BCYE) is required for culture, with up to two weeks of incubation recommended to ensure maximal recovery. Isolates are typically identified using a combination of colony and gram stain morphology, with serologic confirmation and species identification obtained using specific fluorescein-labeled antibodies. Direct detection of organisms in uncultured clinical specimens, usually performed with immunofluorescent methods, is much more rapid than culture but the sensitivity of these methods is poor. A variety of means including radioimmunoassay, enzyme immunoassay, and latex agglutination can be used to detect a soluble polysaccharide antigen of L. pneumophila (serogroup 1 only) in urine with a reported sensitivity of 55-90%. Serological methods are highly sensitive, but their utility is generally limited to epidemiologic studies due to the time lag needed to detect seroconversion. A number of methods have been used in an attempt to identify Legionella organisms in paraffin-embedded tissue sections including various histochemical and immunohistochemical techniques. Silver impregnation stains (e.g., WS staining) serve as the current mainstay of detection in such tissues.
Assays based on molecular diagnostic techniques have included ISH using DNA probes, as well as PCR-based methods. Probes for ISH have largely been directed against rRNA sequences, with sensitivities of approximately 30-75% in both bronchoalveolar lavage (BAL) and fixed tissue specimens. PCR methodology has been used primarily against the 5S and 16S rRNA genes, and against mip of L. pneumophila. The latter amplification assays have been utilized for detection of Legionella species in environmental specimens, serum, urine, throat swabs and BAL specimens resulting in varying degrees of specificity.
Conventional molecular methods used in the above-noted studies require PCR-based amplification followed by detection using probe hybridization, usually on a solid substrate. These methods are labor intensive and frequently require at least one day to perform. Additionally, the required manipulation of post-amplification products increases the risk of carry-over contamination and false-positives. By using commercially available, rapid cycle, real-time PCR instrumentation (e.g., LIGHTCYCLER(trademark), Roche Molecular Biochemicals, Indianapolis, Ind.). PCR amplification and detection can be combined in a single closed cuvette with dramatically reduced cycling time. This method obviates the need for further manipulation of the specimen, greatly reduces turn-around time, and diminishes the risk of cross-contamination between samples. Real-time PCR is an attractive alternative to conventional PCR techniques in the clinical laboratory.
The present invention provides methods for detecting the presence or absence of Legionella in a biological sample from an individual. Methods provided by the invention avoid problems of sample contamination, false-negatives, false-positives, and further allows the specific detection of L. pneumophila. The methods include performing at least one cycling step that includes amplifying and hybridizing. An amplification step includes contacting the sample with a pair of 5S rRNA primers to produce a 5S rRNA amplification product if Legionella 5S rRNA nucleic acid in present in the sample. Each of the 5S rRNA primers anneals to a target within or adjacent to a Legionella 5S rRNA nucleic acid molecule such that at least a portion of the amplification product contains nucleic acid sequence corresponding to 5S rRNA and, more importantly, such that the amplification product contains the nucleic acid sequences that are complementary to 5S rRNA probes. A hybridizing step includes contacting the sample with a pair of 5S rRNA probes. Generally, the members of the pair of 5S rRNA probes hybridize to the amplification product within no more than five nucleotides of each other. According to the invention, a first 5S rRNA probe of a pair of 5S rRNA probes can be labeled with a donor fluorescent moiety and a second 5S rRNA probe of a pair of 5S rRNA probes can be labeled with a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of FRET between the donor fluorescent moiety of the first 5S rRNA probe and the corresponding acceptor fluorescent moiety of the second 5S rRNA probe. Multiple cycling steps can be performed, preferably in a thermocycler. The above-described methods for detecting Legionella in a biological sample using primers and probes directed toward 5S rRNA also can be performed using other Legionella gene-specific primers and probes. In addition, the above-described methods for detecting Legionella in a biological sample using primers and probes directed toward the 5S rRNA also can be performed using mip-specific primers and mip-specific probes to specifically detect L. pneumophila infections.
As used herein, xe2x80x9camplifyingxe2x80x9d refers to the process of synthesizing nucleic acid molecules that are complementary to one or both strands of a template nucleic acid (e.g., Legionella nucleic acid molecules encoding 5S rRNA). Amplifying a nucleic acid molecule typically includes denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature that is below the melting temperatures of the primers, and enzymatically elongating from the primers to generate an amplification product. The denaturing, annealing, and elongating steps each can be performed once. Generally, however, the denaturing, annealing, and elongating steps are performed multiple times such that the amount of amplification product is increasing, oftentimes exponentially, although exponential amplification is not required by the present methods. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g. PLATINUM(copyright) TAQ (derived from recombinant Taq DNA polymerase by binding of a thermolabile inhibitor containing monoclonal antibodies to Taq DNA polymerase such that the inhibitor is denatured during the initial denaturation step of PCR and active Taq DNA polymerase is released into the reaction)) and an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme (e.g. MgCl2 and/or KCl)
If amplification of Legionella nucleic acid occurs and an amplification product is produced, the step of hybridizing results in a detectable signal based on FRET between the members of the pair of probes. As used herein, xe2x80x9chybridizingxe2x80x9d refers to the annealing of probes to an amplification product. Hybridization conditions typically include a temperature that is below the melting temperature of the probes but that avoids non-specific hybridization of the probes.
Generally, the presence of FRET indicates the presence of nucleic acid from Legionella or L. pneumophila in the biological sample and the absence of FRET indicates the absence of Legionella or L. pneumophila nucleic acids in the sample. Inadequate specimen collection, transportation delays, inappropriate transportation conditions, or use of certain collection swabs (calcium alginate or aluminum shaft) are all conditions that can affect the success and/or accuracy of a test result, however. Using the methods disclosed herein, a sample having three times the amount of FRET than that in a control sample lacking Legionella or L. pneumophila nucleic acid generally indicates a Legionella or L. pneumophila infection in the individual.
Representative biological samples that can be used in practicing the methods of the invention include sputum, bronchio-alveolar lavage, bronchial aspirates, lung tissue, urine or blood. Biological sample collection and storage methods are known to those of skill in the art. Biological samples can be processed (e.g., by standard nucleic acid extraction methods and/or using commercial kits) to release Legionella or L. pneumophila nucleic acid or, in some cases, the biological sample is contacted directly with the PCR reaction components and the appropriate oligonucleotides.
Melting curve analysis is an additional step that can be included in a cycling profile. Melting curve analysis is based on the fact that double-stranded nucleic acid xe2x80x9cmeltsxe2x80x9d into single strands at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which half of the DNA duplexes have melted. The melting temperature of a nucleic acid depends primarily upon its nucleotide composition. Thus, nucleic acid molecules rich in G and C nucleotides have a higher Tm than those having an abundance of A and T nucleotides. By detecting the temperature at which signal is lost, the melting temperature of probes can be determined. Similarly, by detecting the temperature at which signal is generated, the annealing temperature of probes can be determined. The melting temperature(s) of the 5S rRNA probes or the mip probes from the respective amplification product can confirm the presence or absence of Legionella or L. pneumophila, respectively, in the sample.
Within each thermocycler run, control samples are cycled as well. Positive control samples can amplify control nucleic acid template (e.g., template other than the 5S rRNA or mip) using, for example, control primers and control probes. Positive control samples can also amplify, for example, a plasmid construct containing Legionella 5S rRNA or L. pneumophila mip nucleic acid. Such a plasmid control can be amplified internally (e.g., within each biological sample) or in separate samples run side-by-side with the patients"" samples. Each thermocycler run should also include a negative control that, for example, lacks Legionella template DNA. Such controls are indicators of the success or failure of the amplification, hybridization, and/or FRET reaction. Therefore, control reactions can readily determine, for example, the ability of primers to anneal with sequence-specificity and to initiate elongation, as well as the ability of probes to hybridize with sequence-specificity and for FRET to occur.
In an embodiment, the methods of the invention include steps to avoid contamination. For example, an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Pat. Nos. 5,035,996, 5,683,896 and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next. In addition, standard laboratory containment practices and procedures are desirable when performing methods of the invention. Containment practices and procedures include, but are not limited to, separate work areas for different steps of a method, containment hoods, barrier filter pipette tips and dedicated air displacement pipettes. Consistent containment practices and procedures by personnel are desirable for accuracy in a diagnostic laboratory handling clinical samples.
Conventional PCR methods in conjunction with FRET technology can be used to practice the methods of the invention. In one embodiment, a LIGHTCYCLER(trademark) instrument is used. A detailed description of the LIGHTCYCLER(trademark) System and real-time and on-line monitoring of PCR can be found on Roche""s website. The following patent applications describe real-time PCR as used in the LIGHTCYCLER(trademark) technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LIGHTCYCLER(trademark) instrument is a rapid thermocycler combined with a microvolume fluorimeter utilizing high quality optics. This rapid thermocycling technique uses thin glass cuvettes as reaction vessels. Heating and cooling of the reaction chamber are controlled by alternating heated and ambient air. Due to the low mass of air and the high ratio of surface area to volume of the cuvettes, very rapid temperature exchange rates can be achieved within the LIGHTCYCLER(trademark) thermal chamber. Addition of selected fluorescent dyes to the reaction components allows the PCR to be monitored in real-time and on-line. Furthermore, the cuvettes serve as an optical element for signal collection (similar to glass fiber optics), concentrating the signal at the tip of the cuvette. The effect is efficient illumination and fluorescent monitoring of microvolume samples.
The LIGHTCYCLER(trademark) carousel that houses the cuvettes can be removed from the instrument. Therefore, samples can be loaded outside of the instrument (in a PCR Clean Room, for example). In addition, this feature allows for the sample carousel to be easily cleaned and sterilized. The fluorimeter, as part of the LIGHTCYCLER(trademark) apparatus, houses the light source. The emitted light is filtered and focused by an epi-illumination lens onto the top of the cuvette. Fluorescent light emitted from the sample is then focused by the same lens, passed through a dichroic mirror, filtered appropriately, and focused onto data-collecting photohybrids. The optical unit currently available in the LIGHTCYCLER(trademark) instrument (Roche Molecular Biochemicals, Catalog No. 2 011 468) includes three band-pass filters (530 nm, 640 nm, and 710 nm), providing three-color detection and several fluorescence acquisition options. The present invention, however, is not limited by the configuration of a commercially available instrument. Data collection Options include once per cycling step monitoring, fully continuous single-sample acquisition for melting curve analysis, continuous sampling (in which sampling frequency is dependent on sample number) and/or stepwise measurement of all samples after defined temperature interval.
The LIGHTCYCLER(trademark) can be operated using a PC workstation and can utilize a Windows NT operating system. Signals from the samples are obtained as the machine positions the capillaries sequentially over the optical unit. The software can display the fluorescence signals in real-time immediately after each measurement. Fluorescent acquisition time is 10-100 msec. After each cycling step, a quantitative display of fluorescence vs. cycle number can be continually updated for all samples. The data generated can be stored for further analysis.
A common FRET technology format utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and the two probes are generally designed to hybridize in close proximity to each other in a target DNA molecule (e.g., an amplification product). By way of example, a donor fluorescent moiety such as fluorescein can be excited at 470 nm by the light source of the LiGHTCYCLER(trademark) Instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety such as LC(trademark)-RED 640 (LIGHTCYCLER(trademark)-Red 640-N-hydroxysuccinimide ester) or LCW(trademark) RED 705 (LightCycler(trademark)-Red 705-Phosphoramidite). The acceptor fluorescent moiety then emits light of a longer wavelength (e.g., 640 nm or 705 nm, respectively), which is detected by the optical detection system of the LIGHTCYCLER(trademark) instrument. Other donor and corresponding acceptor fluorescent moieties suitable for use in the invention are described above. Efficient FRET can only take place when the fluorescent moieties are in direct local proximity (for example, within 5 nucleotides of each other as discussed above) and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can bc correlated with the number of original target nucleic acid molecules (e.g., the number of Legionella or L. pneumophila organisms).
Another FRET technology format utilizes TAQMAN(copyright) technology to detect the presence or absence of an amplification product, and hence, the presence or absence of Legionella. TAQMAN(copyright) technology utilizes one single-stranded hybridization probe labeled with two fluorescent moieties. When a first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to a second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is generally a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is degraded by the 5xe2x80x2 to 3xe2x80x2 exonuclease activity of the Taq Polymerase during the subsequent elongation phase. As a result, the excited fluorescent moiety and the quencher moiety become spatially separated from one another. As a consequence, upon excitation of the first fluorescent moiety in the absence of the quencher, the fluorescence emission from the first fluorescent moiety can be detected. By way of example, an ABI PRISM(copyright) 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.) uses TAQMAN(copyright) technology, and is suitable for performing the methods described herein for detecting Legionella. Information on PCR amplification and detection using an ABI PRISM(copyright) 770 system can be found on Applied Biosystems"" website.
Yet another FRET technology format utilizes molecular beacon technology to detect the presence or absence of an amplification product, and hence, the presence or absence of Legionella. Molecular beacon technology uses a hybridization probe labeled with a donor fluorescent moiety and an acceptor fluorescent moiety. The acceptor fluorescent moiety is generally a quencher, and the fluorescent labels are typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide having sequences that permit secondary structure formation (e.g., a hairpin). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. After hybridization to the target nucleic acids (i.e., the amplification products), the secondary structure of the probe is disrupted and the fluorescent moieties become separated from one another such that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.
As an alternative to detection using FRET technology, an amplification product can be detected using a nucleic acid binding dye such as a fluorescent DNA binding dye (e.g., SYBROREENI(copyright) or SYBRGOLD(copyright) (Molecular Probes)). Upon interaction with the double-stranded nucleic acid, such nucleic acid binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength. A nucleic acid binding dye such as a nucleic acid intercalating dye also can be used. When nucleic acid binding dyes are used, a melting curve analysis is usually performed for confirmation of the presence of the amplification product.
It is understood that the present invention is not limited by the configuration of one or more commercially available instruments.
Articles of Manufacture
The invention further provides for articles of manufacture to detect Legionella and specifically L. pneumophila. An article of manufacture according to the present invention can include primers and probes used to detect Legionella, together with sutiable packaging material. Representative primers and probes provided in a kit for detection of Legionella can be complementary to Legionella nucleic acid molecules encoding 5S rRNA. Similarly, representative primers and probes for detection of L. pneumophila can be complementary to L. pneumophila nucleic acid molecules encoding mip. Methods of designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to Legionella nucleic acids encoding 5S rRNA or mip are provided.
Articles of manufacture of the invention also can include one or more fluorescent moieties for labeling the probes or, alternatively, the probes supplied with the kit can be labeled. For example, an article of manufacture may include a donor fluorescent moiety for labeling one of the 5S rRNA or mip probes and a corresponding acceptor fluorescent moiety for labeling the other 5S rRNA or mip probe. Examples of suitable FRET donor fluorescent moieties and acceptor fluorescent moieties are provided herein.
Articles of manufacture of the invention also can contain a package insert having instructions thereon for using pairs of 5S rRNA primers and 5S rRNA probes to detect Legionella in a biological sample. Such a package insert may contain instructions thereon for using pairs of mip primers and mip probes to specifically detect L. pneumophila in a biological sample. Articles of manufacture may additionally include reagents for carrying out the methods disclosed herein (e.g., buffers, polymerase enzymes, co-factors, or agents to prevent contamination). Such reagents may be specific for one of the commercially available instruments described herein.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.